Construction

From 2006.igem.org

Latest revision as of 08:14, 2 November 2006

October 30, 2006

Charles:

• We sent in our parts to MIT!
• Re mini-prepped UT9 ABCDE and digested using XbaI/PstI
• UT9 ABCDE – (2500 – 2000, 1000 – 750) – Correct!
• Gel extracted and quantitated: got no bands =(
• Made Freezer stock of DH5a and DH5a-z1
• Made o/n of UT14 and pBAD for mini-prep, DH5a and DH5a-z1 for competent cells
• Tested Module 2 with constant 0.2% arabinose and increasing aTc from 0, 5, 10, 20, 50 ng/mL for both UT10 and DH5a
• Made o/n of UT2/UT3 and UT10/UT11 for possible testing, UT9 BCDEFG for miniprep and gel extract

To-Do List:

• Make more M9 media
• Make competent cells
• Ligate final parts together

October 29, 2006

Charles:

• Gel extracted:
  • UT10 A = 14 ng/uL
  • UT10 B = 16 ng/uL
  • UT10 C = 14 ng/uL
  • UT11 ABC ~ 0 
• Made o/n of DH5a and DH5a-z1, as well as UT10/UT11 ABC for aTc testing
• Mini-prepped UT9 DE, UT14 AB, pBAD 33 AB
• Length check: (none worked)
  • UT9 DE (S/P), UT14 AB (E/S), pBAD A (S/P) -- none worked
• made more o/n of UT9 ABCDE and UT10/UT11 DE

October 28, 2006

Charles:

• Mini-prepped UT9, UT10, UT11 ABC (may be slightly messed up – miniprep again?)
• Length check:
  • I0500 – no bands =(
  • Q04400 (2005/2006) – correct
  • Q03400 - correct
  • UT9 – no bands =( try again tomorrow with concentrated DNA
  • UT10 - correct
  • UT11 - correct
• UT9 looks red
• Tested UT10/11 under +aTc = 50 ng/mL and –aTc = 0 ng/mL conditions
• Plated freezer stock of DH5a and DH5a-z1 to make a new plate
• Made o/n of pBAD AB and UT9 DEF for miniprep

To-Do List: - Make o/n of DH5a and DH5a-z1 - Miniprep pBAD AB - Make Amp and Kan plates

October 27, 2006

Charles, Nick:

• M9 Test – 0% - 2% Arabinose in UT3 and DH5a (control), temperature test at 0.2% Arabinose, IPTG verification at 0% and 2% arabinose:
  • Dilute o/n of UT3 and DH5a in 1:50 o/n to LB ratio in appropriate arabinose/IPTG concentrations – incubate 3 hrs
  • Resuspend in 50:50 M9:LB (including arabinose/IPTG) and divided 0.2% arabinose for temperature test – incubate for 3 hrs
  • Resuspend in M9 media – incubate for 5 hrs
    • First 3 hrs without signalling conditions, then latter 2 hrs with conditions
  • Read with fluorometer (PBS wash not required)
• Ligated I0500 B (2005) + I13507 (2005) / I13504 (2005/2006)
• Made o/n of UT9, UT10, UT11 ABC
• Made o/n of DH5a and DH5a-z1

To-Do List:

• Check lengths of I0500 (2006), Q04400 (2005/2006), Q03400 (2006)
• Miniprep and check lengths of UT9, UT10, UT11 – if correct, wait for functionality test before transforming into cells
• Make competent cells (DH5a and DH5a-z1)
• Make Amp and Kan plates

October 26, 2006

Andy, Natalie, Charles, Tara, HoKwon:

• Ligated and transformed UT6, UT7 with I13504 (2005/2006) (Name UT10, UT11) (Quantitation showed correct band for all)
• Ligated and transformed R0011 (2005) with I13507 (2005) (Name UT9) (Quantitation showed correct band for all)
• Made o/n of UT3 x 3, DH5a for testing
• Quantitated I0500 (2005) AB to be ligated to I13504 (2005/2006) (Band: 6000 - roughly correct. A 20 ng/uL B 300 ng/uL)

To-do list:

• Make o/n of UT9, UT10, UT11 for miniprep
• Ligate I0500 (2005) with I13504 (2005/2006)
• M9 testing of UT3

October 25, 2006

Andy:

• Minipreppred UT6, UT7, UT8
• Gel-extracted (only for correct bands)
  • UT6 S/P (band: 8000-10000 correct) -- 80 ng/uL
  • UT7 S/P (band: 8000-10000 correct) -- 50 ng/uL
  • UT8 S/P (band: 6000 incorrect)
  • I13504 (2005) X/P (band: 750-1000, 1500-2000 correct) -- 140 ng/uL
  • I13504 (2006) X/P (band: 750-1000, 1500-2000 correct) -- 90 ng/uL
• Made o/n of UT2 x 3, UT3 x 3, DH5a for testing

To-do list:

• Quantitate and ligate UT6, UT7 with I13504 (2005/2006)
• Quantitate and ligate R0011 (2005) with I13507 (2005)
• M9 testing of UT2, UT3

October 24, 2006

Melinda:

• Minipreppred and length-checked (unsuccessful) I13522 (2006), I13504 (2005/2006) AB, R0011 (2005)
• Made o/n of UT6-8 for miniprep

October 23, 2006

Andy:

• Gel-extrated I13507 (2005) X/P
• Made o/n of I13522 (2006), I13504 (2005/2006) x 2, R0011 (2005) for minipreps
• Tested UT3 for temperature and arabinose sensitivity, plated UT2, UT3, DH5a for plate test
• Made plates
• ligated and transformed UT1 with Q4400 (2005) = UT6, Q3400 (2006) = UT7, and Q4400 (2006) = UT8

To-do List:

• Quantitate and Ligate R0011 (2005) with I13507 (2005)
• Miniprep and length-check I13522 (2006), I13504 (2005/2006) x 2, R0011 (2005)
• Make multiple o/n of UT6-8 for miniprep

October 22, 2006

Massive To-do List

The following is a guideline. Day-to-day details need to be filled in as we progress. Change colors from red to black as each task is completed.

Construction:

• Transform and miniprep I13522 (2006) (amp) from registry DNA-2 15H
• Transform and miniprep I13504 (2005/2006) (amp) from registry DNA-1 12D (2005/2006)
• Miniprep 2 UT1 for ligation (not urgent)
• Ligate UT1 with Q03400, Q04400 (2005/2006) (4 ligations) (Name UT6-9)
• If possible, ligate R0011 (2005) with I13507 (2005) in parallel (Name UT10)
• Ligate UT6-9 with I13504 (4 ligations) (Name UT11-14)
• Ligate UT11-14 with UT10 (4 ligations) (Nam UT15-18)

Testing:

• Temperature-test and Arabinose/IPTG-test UT3 (See below for protocol)
• Plate-test UT2, UT3, DH5a, UT13, I13522 (See below for protocol)

Andy:

• Gel-extrated R0011 (2005) S/P (band: 2000 - correct)
• Cannot gel-extract I13507 (2005) X/P because there was no more extraction column (bands: 2500-3000, 2000-2500, 750-1000 - correct)
• Transformed R0011 (2005), I13522 (2006), I13504 (2005/2006)
• Quantitated
  • Q3400 (2005) E/X (no band)
  • Q4400 (2005) E/X (band: 5000 - correct 120 ng/uL)
  • Q3400 (2006) E/X (band: 5000 - correct 24 ng/uL)
  • Q4400 (2006) E/X (band: 5000 - correct 120 ng/uL)
  • UT1 D E/S (band: 2500-3000 - correct 18 ng/uL)
  • UT1 E E/S (band: 2500-3000 - correct 12 ng/uL)
  • UT1 F E/S (band: 2500-3000 - correct 30 ng/uL)
• o/n of UT3 x 5, UT2 x 1, and DH5a x 4 for temperature, arabinose, and plate testing

To-do List:

• Ligate UT1 with Q3400 (2006), Q4400 (2005/2006)
• Make o/n of I13522 (2006) for miniprep and plate-test, I13504 (2005/2006), I13507 (2005) for miniprep
• Gel-extract I13507 (2005) X/P
• Temperature, arabinose, and plate testing
• Purchase gel-extraction kit, make Amp plates

Guidelines for Plate-test:

1. On an Amp plate, make 4 sectors (500 uL?) with varying arabinose (0.02%, 0.2%, 2%, 20%) and making sure they don’t touch each other (otherwise diffusion will happen and they’ll be the same concentration), let the arabinose area dry.
2. Take o/n of UT3-DH5a and take out 200 uL and add 800 uL of fresh LB and incubate for 1 hr.
3. Spin down the cells at 3000 RPM for 5 min and remove 800 uL of supernatant. Resuspend cells with the remaining 200 uL and gently spread onto plate

Guidelines for Temperature-test:

1. Put a constant amount of arabinose (either 0.02% or 0.2% - don’t want TOO much repression) in each dilution.
2. Grow dilutions at 37C until the absorbance is ~0.6 using the GFP ABS program. Then take some tubes out and put 1 mL into an Eppendorf tube.
3. Use the white filter caps and put them on each Eppendorf tube (to allow air in, but to protect against contamination)
4. Set mixer to the equivalent to the shaker in the incubator (remember it’s a smaller tube, so there probably needs to be more force on it to get it to shake).
5. Set temperature to 22C
6. Check the absorbance after a couple of hours and prepare the samples when the absorbance matches the absorbance of the tubs in the incubator (these should be ready before the 22C ones are ready)
7. Hopefully we’ll see a significant difference in fluorescence: cold should fluoresce less than hot.

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October 19, 2006

Melinda:

• Miniprepped Q04400 (2005/2006) and Q04510 (2005/2006)
• Made o/n of Q03400 (2005/2006), (3) UT2-DH5a, (2) DH5a, and (1) DH5a/DH5a-z1 for freezer stock and fresh plate

To-Do List:

• Miniprep Q03400 (2005/2006)
• Repeat Oct 18 test, except with GFP and proper controls
• Digest and check J04450 (W), Q03400, Q04400, and Q04510 (2005/2006). If correct, move on with gel extraction and ligation.
• Make 20% Arabinose, and 2 blank agar plates

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October 18, 2006

Andy, Konstantin:

• Transformed and plated Q03400 (2005) (2006)
• Gel-extracted UT1 DEF with EcoRI/SpeI (2500-3000, 5000, 8000), 8000 band bright, digestion appeared incomplete
• Made A, K, AK plates
• Made o/n of Q04400 (2005) (2006) Q04510 (2005) (2006)
• Tested UT3 in DH5a with 0%-2% arabinose

To-Do List:

• Make o/n of Q03400 (2005) (2006)
• Miniprep Q04400 (2005) (2006) and Q04510 (2005) (2006)
• Replate UT1 with miniprep from a good conlony
• Miniprep Q04400 (2005) (2006) Q04510 (2005) (2006) and check to see if bands are correct. If so, digest with EcoRI/XbaI and gel extract
• Ligate with UT1

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October 17, 2006

Melinda:

• Double Digetsed J04450 (W), UT1 DEFG with XbaI/SpeI using Buffer 2
• Results:
  • J04450 (W) (1 band)
  • UT1 D (5000 – 4000, 3000 – 2500)
  • UT1 E (2 bands)
  • UT1 F (2 bands)
  • UT1 G (2 bands)
• Transformed DH5a cells with Q04400 (2005/2006), Q04510 (2005/2006)
• Made o/n of DH5a and (3 vials) of DH5a UT3

Note – Change in protocol:

• After incubation for 1 hour, centrifuge tubes for 5 min @ 3000 RPM to pellet cells
• Remove 800 uL of supernatant
• Resuspend pellet with remaining 200 uL
• Spread on plate and wait ~15-20 min.

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October 16, 2006

Melinda:

• Checked parts, including the relevant parts from Waterloo (W):
  • I0500 (W): (5000 – 4000, 1500 – 1000) – correct!
  • J04450 (W): (2500 – 2000) – plasmid is correct, but no part?
  • J06801 (W): (8000 – 6000) – not correct!
  • E0240 (W): (4000 – 3500, 3500 – 3000, 1000 – 750) – part is correct?
  • I12006 (W): (5000 – 4000, 2500 – 2000) - plasmid is correct, undigested plasmid?
  • UT2 2: (3500 – 3000, 2500 – 2000, 2000 – 1500) – correct, extra band?
  • UT2 4: (3500 – 3000, 2500 – 2000, 2000 – 1500) – correct, extra band?
  • UT3 7: (3500 – 3000, 2500 – 2000) – correct!
• Viewed cells induced by IPTG under microscope and verified the functionality of UT2/UT3 in DH5a and DH5a-z1

To-Do List:

• Repeat Double Digest of J04450 (W) and UT1 ABCD with XbaI/SpeI (Run undigested plasmids beside each double digest)
• Make more Amp plates
• If digest is successful, transform DH5a cells with the successful parts
• Find tetR replacements for cI and ligate those parts together.

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October 15, 2006

Charles:

• Made o/n of the newly plated UT2 2/4 and UT3 7 as well as UT2/UT3 freezer stock

To-Do List:

• Make sure the cells fluoresce by diluting in IPTG

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October 14, 2006

Charles, Jovan:

• Mini-prepped UT2 2/4 and UT3 7
• Transform UT2 2/4 and UT3 7 into DH5a-z1 and DH5a cells
• Continue with the IPTG test for the rest of the colonies that we weren’t able to test yesterday

To-Do List:

• Measure absorbance spectrum of Arabinose
• Make o/n of working UT2/UT3 for repeat of IPTG test.
• Look into using tetR and tet pL instead of cI and Prm+

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